Drug resistant immunotherapy for treatment of a cancer

ABSTRACT

The present disclosure is generally related to methods for combining chemotherapy and immunotherapy for the treatment of a cancer. The methods also relate to generating a drug-resistant cytotoxic immune cell line and uses thereof in conjunction with cytotoxic drugs.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. provisional application No.61/257,136, filed on Nov. 2, 2009, which is hereby incorporated byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under NIH Grant No. HL087969-01A1 awarded by the U.S. National Institutes of Health of theUnited States government. The government has certain rights in theinvention

TECHNICAL FIELD

The present disclosure is generally related to methods for combiningchemotherapy and immunotherapy for the treatment of a cancer. Themethods also relate to generating a drug-resistant cytotoxic immune cellline and uses thereof in conjunction with cytotoxic drugs.

BACKGROUND

Although outstanding progress has been made in the fields of cancerdetection and tumor cell biology, the treatment of late-stage andmetastatic cancer remains a major challenge. Cytotoxic chemotherapyagents remain among the most used and successfully employed anti-cancertreatments. However, they are not uniformly effective, and theintroduction of these agents with novel therapies, such asimmunotherapies, is problematic. For example, chemotherapy agents can bedetrimental to the establishment of robust anti-tumor immunocompetentcells due to the agents' non-specific toxicity profiles. Smallmolecule-based therapies targeting cell proliferation pathways may alsohamper the establishment of anti-tumor immunity. However, ifchemotherapy regimens that are transiently effective can be combinedwith novel immunocompetent cell therapies then significant improvementin anti-neoplastic therapy might be achieved.

Several drug resistant genes have been identified that can potentiallybe used to confer drug resistance to targeted cells, and advances ingene therapy techniques have made it possible to test the feasibility ofusing these genes in drug resistance gene therapy studies (Sugimoto etal., (2003) J. Gene Med. 5: 366-376; Spencer et al., (1996) Blood 87:2579-2587; Takebe et al., (2001) Mol. Ther. 3: 88-96; Kushman et al.,(2007) Carcinogenesis. 28: 207-214; Nivens et al., (2004) CancerChemother. Pharmacol. 53: 107-115; Bardenheuer et al., (2005) Leukemia19: 2281-2288; Zielske et al, (2003) J. Clin. Invest. 112: 1561-1570).For example, a shRNA strategy was used to decrease the levels ofhypoxanthine-guanine phosphoribosyltransferase (HPRT), which conferredresistance to 6-thioquanine (Porter & DeGregori (2008) Gene Ther. 112:4466-4474). Also, the drug resistant gene MGMT encoding human alkylguanine transferase (hAGT) is a DNA repair protein that confersresistance to the cytotoxic effects of alkylating agents, such asnitrosoureas and temozolomide (TMZ). 6-benzylguanine (6-BG) is aninhibitor of AGT that potentiates nitrosourea toxicity and isco-administered with TMZ to potentiate the cytotoxic effects of thisagent. Several mutant forms of MGMT that encode variants of AGT arehighly resistant to inactivation by 6-BG, but retain their ability torepair DNA damage (Maze et al., (1999) J. Pharmacol. Exp. Ther. 290:1467-1474). P140KMGMT-based drug resistant gene therapy has been shownto confer chemoprotection to mouse, canine, rhesus macaques, and humancells, specifically hematopoetic cells (Zielske et al, (2003) J. Clin.Invest. 112: 1561-1570; Pollok et al., (2003) Hum. Gene Ther. 14:1703-1714; Gerull et al, (2007) Hum. Gene Ther. 18: 451-456; Neff etal., (2005) Blood 105: 997-1002; Larochelle et al., (2009) J. Clin.Invest. 119: 1952-1963; Sawai et al., (2001) Mol. Ther. 3: 78-87).

Glioblastoma multiforme (GBM) is the most common and most aggressivetype of primary brain tumor in humans, involving glial cells andaccounting for 52% of all parenchymal brain tumor cases and 20% of allintracranial tumors. Despite being the most prevalent form of primarybrain tumor, GBMs occur in only 2-3 cases per 100,000 people in Europeand North America. The standard name for this brain tumor is“glioblastoma”; it presents two variants: giant cell glioblastoma andgliosarcoma. Glioblastomas are also an important brain tumor of thecanine, and research is ongoing to use this as a model for developingtreatments in humans.

Glioblastoma has one of the poorest prognoses among the cancers.Treatment can involve chemotherapy, radiation and surgery, alone or incombination, but the outcome is still typically unfavorable for thepatient. For example, the median survival with standard-of-careradiation and chemotherapy with temozolomide is just 15 months. Mediansurvival without treatment is about four and one-half months. Thereremains, therefore, an urgent need for methods that enhance, replace orsupplement current methods of treating such cancers, and in particularthose that exhibit transient responses to chemotherapy. Immunotherapyoffers such a supplemental procedure if the cytotoxicity of thechemoagent can be circumvented.

SUMMARY

Establishment of immunocompetent cell mediated anti-tumor immunity isoften mitigated by the myelosuppressive effects during chemotherapy. Thepresent disclosure provides methods for protecting these immune cellsfrom drug induced toxicities, thereby allowing for the combinedadministration of immuno- and chemotherapy, an anticancer treatmenttermed “drug resistant immunotherapy”. Using a SIV-based lentiviralsystem, the drug resistance-conferring genetic element can be deliveredinto immunocompetent cell lines. Genetically engineered immunocompetentcells developed significant resistance to a specific chemotherapeuticcytotoxic agent compared to non-modified cells, and did not affect theirability to kill target cancer cells in the presence or absence of achemotherapy agent. Engineering immunocompetent cells to withstandchemotherapy challenges can enhance tumor cell killing when chemotherapyis applied in conjunction with cell-based immunotherapy.

One aspect of the present disclosure, therefore, encompasses methods forreducing a cancer in a patient, comprising the steps of: obtaining apopulation of isolated cytotoxic immune cells, where the isolatedcytotoxic immune cells have been genetically modified to be resistant toa therapeutic agent; administering to a patient in need thereof, aneffective amount of the therapeutic agent; and administering to thepatient population of isolated genetically modified cytotoxic immunecells, whereupon the cytotoxic immune cells are delivered to the tumor,thereby reducing the cancer in the patient.

In embodiments of this aspect of the disclosure, the isolated cytotoxicimmune cells can be γδ T-cells.

In embodiments of this aspect of the disclosure, the step of obtaining apopulation of isolated cytotoxic immune cells genetically modified to beresistant to a therapeutic agent can comprise: isolating from a subjecthuman or animal a population of cytotoxic immune cells; culturing theisolated population of cytotoxic immune cells, thereby increasing thepopulation of the cells; stably transfecting the population of cytotoxicimmune cells with a vector comprising a heterologous nucleic acidsequence operably linked to a promoter, wherein the heterologous nucleicacid sequence encodes a polypeptide conferring to the cell resistance tothe therapeutic agent.

Yet another aspect of the present disclosure provides systems fortreating a cancer in a patient comprising a cytotoxic therapeutic agenthaving the characteristics of inhibiting the survival of a cancer cell,and an isolated population of cytotoxic immune cells, where thecytotoxic immune cells genetically modified to be resistant to thetherapeutic agent.

Still another aspect of the disclosure provides systems for treating aglioblastoma in a patient comprising a therapeutic agent having thecharacteristics of inhibiting the survival of a cancer cell and inducinga stress protein in the cancer cell, and an isolated population ofcytotoxic immune cells, wherein said cytotoxic immune cells are γδT-cells, and wherein said γδ T-cells have been genetically modified tobe resistant to the therapeutic agent.

In certain embodiments, the invention relates to methods of treating asubject diagnosed with cancer comprising administering a chemotherapyagent to the subject and administering a chemotherapy resistant cellcomposition to the subject wherein the chemotherapy resistant cellcomposition comprises cells genetically engineering to express apolypeptide that confers resistance to the chemotherapy agent.

In certain embodiments, the invention relates to isolated compositionscomprising natural killer cells wherein greater than about 50%, 60%, 70%80%, 90%, or 95% of the natural killer cells express a polypeptide thatconfers resistance to a chemotherapy agent or isolated compositionscomprising natural killer cells wherein greater than about 50%, 60%, 70%80%, 90%, or 95% of the natural killer cells comprise a nucleic acidthat encodes a polypeptide that confers resistance to a chemotherapyagent or isolated compositions consisting essentially of natural killercells comprising a nucleic acid that encodes a polypeptide that confersresistance to a chemotherapy agent. In further embodiments, thepolypeptide that confers resistance to a chemotherapy agent is 06methylguanine DNA methyltransferase (MGMT), a drug resistant variant ofdihydrofolate reductase (L22Y-DHFR), thymidylate synthase, and/ormultiple drug resistance-1 protein (MDR1).

BRIEF DESCRIPTION OF THE DRAWINGS

Many aspects of the disclosure can be better understood with referenceto the following drawings.

FIG. 1 schematically compares a protocol for combining immunotherapy andchemotherapy in the treatment of a cancer, where the immune cells aresensitive (top) and resistant (bottom) to the chemotherapeutic agent. Inthe non-resistant scheme, an anti-tumor response is provided bycytokines such as IL-2, IL-12, GM-CSF, and the like.

FIGS. 2A and 2B are graphs showing that γδ cells kill glioblastoma celllines in a dose response fashion (FIG. 2A), and the viability of theglioblastoma cells decrease with increasing amounts of γδ cells (FIG.2B).

FIG. 2C shows a series of digital images showing the killing ofglioblastoma cells.

FIG. 3 shows a bar graph illustrating the cytotoxicity of isolatedγδT-cells against several cultured glioblastoma cell isolates,including: cultured primary (GBM 1 and GBM 2) glioblastomal cells andcultured cell lines D54MG, U251MG, and U373.

FIG. 4 shows a pair of bar graphs illustrating that cells involved inthe adaptive immune response are sensitive to current glioblastomatreatment regimens (left). γδ cells are similarly sensitive (right).

FIG. 5 is a graph showing the expression of cell surface stress antigensMICA/B induced by the chemotherapeutic agent Temozolamide (TMZ).

FIG. 6 is a graph showing that the expression of cell surface stressantigens MICA/B induced by the chemotherapeutic agent Temozolamide (TMZ)is transient.

FIG. 7 shows a series of digital images of the development ofglioblastomas in mice injected with saline (top) and γδ T-cells(bottom).

FIG. 8 is a graph showing the imaging density in mice 1-3 weeks aftertumor induction and treatment with saline (black circles) or γδ T-cells(open circles).

FIG. 9 is a graph showing the increased survival of mice having inducedglioblastomas following γδ T-cell treatment (open circles).

FIG. 10 schematically compares a protocol for combining immunotherapyusing γδ T-cells and chemotherapy in the treatment of a cancer, wherethe γδ immune cells are sensitive (top) and resistant (bottom) to thechemotherapeutic agent.

FIG. 11 is a graph illustrating that transduction of fibroblasts with aheterologous nucleic acid sequence encoding MGMT confers resistance tothe compound BCNU (bis-chloronitrosourea; CARMUSTINE™), a mustardgas-related α-chloro-nitrosourea compound used as an alkylating agent inchemotherapy, particularly for treatment of glioblastomas.

FIG. 12 schematically shows the region of a lentivirus that includes twolong terminal repeats (LTR) and a heterologous nucleic acid sequenceencoding a green fluorescent protein (GFP) or MGMT variant P104K.

FIG. 13 is a series of digital photographs illustrating transduction ofγδT-cells with SIV-GFP or SIV-MGMT constructs. The top panels show hightransduction efficiency using a GFP-encoding construct. As expected, nofluorescence is observed with MGMT (bottom panel).

FIG. 14 is a graph showing that the administration of a chemotherapeuticagent to an anti-cancer regimen, which also requires T-cell expansion,decreases the effectiveness of the cell-based treatment. This decreaseis very pronounced when the mice have undergone a bone marrow transplantprocedure, which is a common procedure for patients undergoing treatmentfor several types of cancer.

FIG. 15 schematically shows an experimental protocol for treating acancer with drug resistant immune cells. In this protocol, bone marrowwas harvested from mice and transduced with a recombinant lentivirusvector comprising the heterologous nucleic acid sequence encoding theL22YDHFR variant. The transduced cells were transplanted into irradiatedrecipient mice. After 4 weeks AG104 sarcoma cells were transplanted. Twoweeks later the mice would receive immunotherapy comprising anti-CD137antibodies followed by chemotherapy (TMTX).

FIG. 16 shows a pair of graphs illustrating the effect of chemotherapy(TMTX) treatment alone or immunotherapy alone (anti-CD137 antibodystimulation of cytotoxic lymphocytes). In each case the protocol schemeshown in FIG. 15 was followed.

FIG. 17 shows a graph illustrating the rapid and prolonged reduction inAG104 tumor size with the immunotherapy-chemotherapy combination wherethe immune system of the tumor-bearing mice was rendered TMTX-resistantby transduction with L22YDHFR.

FIG. 18 shows a survival chart for DHFR-bearing mice transplanted withAG104 sarcoma cells and treated with TMTX and/or anti-CD137immunotherapy.

FIG. 19 shows a survival chart for mice receiving splenocytes isolatedfrom tumor-bearing and DHFR-cured mice and then challenged with AG104sarcoma cells.

FIG. 20A and FIG. 20B show data suggesting genetically engineered γδ Tcells killed both wt and TMZ resistant GBM cells in the presence ofagent. Non-modified γδ T cells are not active in the presence of TMZ. Emis gene-modified γδ T cells. E is non-modified γδ T cells. T is SB19 TMZresistant.

FIG. 21 shows data suggesting genetic modification of γδ T cells confersresistance to TMZ. γδ T cells were transduced (MOI=20) withSIV-MGMT-DHFR at day 8 of expansion and cell viability was measured atday 14. Viability was assessed by uptake of ToPro Iodide.

FIG. 22 shows data suggesting genetically engineered γδ T cells retainedtheir cytotoxicities towards GBM cells. E is Wild type γδ T cells; Em isgene-modified γδ T cells

T is SB19 (GBM) cell lines; (four-hour cytotoxicity assay).

FIG. 23 shows a molecular model of DHFR and the locations of variantsites. Also shown is a graph showing the effectiveness of the variantsin conferring drug resistance to cells transfected and expressing thevariant polypeptides.

FIGS. 24A-24D are a series of graphs that illustrate the determinationof transduction efficiencies for immunocompetent and experimental targetcells.

FIG. 24A illustrates schematics of SIV vector constructs encoding foreGFP (top) and P140KMGMT (bottom).

FIG. 24B is an image of a flow cytometry analysis of NK-92 cellstransduced with SIV-eGFP lentivirus.

FIG. 24C is an image of a flow cytometry analysis of TALL-104 cellstransduced with SIV-eGFP lentivirus.

FIG. 24D is an image of a flow cytometry analysis of K562 cellstransduced with SIV-eGFP lentivirus.

FIG. 25A is a series of graphs showing the survival curve analyses ofP140KMGMT-modified (open circles) and non-modified (closed circles)NK-92 cells (left panel), TALL-104 cells (middle panel) and K562 cells(right panel) cells after 6-BG/TMZ treatment. The cells were treatedwith 25 μM 6-BG and increasing concentrations of TMZ. Forty eight hourslater, cell viabilities were measured by a trypan blue method. Each datapoint in all the graphs represents the mean of triplicate values.

FIG. 25B is a pair of graphs showing the cytotoxic activities of theimmune effector NK-92 cells (left panel) and TALL-104 cells (rightpanel) against K562 target cells at different effector:target (E:T) cellratios. Different concentrations of P140KMGMT-modified cells (opencircles), gene-modified cells after selection with 25 μM 6-BG/200 μM TMZ(reverse triangle), or non-modified cells (closed circles) were mixedwith a fixed concentration of the target cells and LDH release assayswere performed after 4 hours. Each data point in all the graphsrepresents the mean of triplicate values.

FIG. 26 is a pair of graphs showing non-engineered immune effectorcell-mediated lysis of the target K562 cells. Non-modified (E) effectorcells, and non-modified (T) or gene-modified (Tm) target K562 cells weretreated with 25 μM 6-BG/200 μM TMZ overnight. The non-modified (E)effector cells were then mixed with either non-modified (T) orgene-modified (Tm) target K562 cells at an E:T ratio of 10:1. Thecytotoxic activities of the effector cells were then measured. Panel Arepresents NK-92 cell-mediated lysis; Panel B represents TALL-104cell-mediated lysis.

FIGS. 27A-27D are a series of graphs illustrating genetically engineeredimmune effector cell mediated lysis of the target K562 cells in thepresence of 6-BG/TMZ. The non-modified (E) and gene-modified (Em)effector cells, and the non-modified (T) or gene-modified (Tm) targetcells were treated with 25 μM 6-BG/200 μM TMZ overnight. Thenon-modified or modified effector cells were incubated withgene-modified target cells at an E:T ratio of 10:1, and cytotoxicactivities of the effector cells were measured. Different combinationsof either non-modified or gene-modified effector cells were mixed witheither non-modified or gene-modified target cells at an E:T ratio of10:1. Cytotoxicities of the effector cells were determined.

FIG. 28 shows the nucleotide sequences encoding MGMT and DHFR codonoptimized for expression in mammalian cells.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit (unlessthe context clearly dictates otherwise), between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of chemistry, synthetic organic chemistry,biochemistry, biology, molecular biology, molecular imaging, and thelike, which are within the skill of the art. Such techniques areexplained fully in the literature.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how toperform the methods and use the compositions and compounds disclosed andclaimed herein. Efforts have been made to ensure accuracy with respectto numbers (e.g., amounts, temperature, etc.), but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C., and pressure is at or nearatmospheric. Standard temperature and pressure are defined as 20° C. and1 atmosphere.

Before the embodiments of the present disclosure are described indetail, it is to be understood that, unless otherwise indicated, thepresent disclosure is not limited to particular materials, reagents,reaction materials, manufacturing processes, or the like, as such canvary. It is also to be understood that the terminology used herein isfor purposes of describing particular embodiments only, and is notintended to be limiting. It is also possible in the present disclosurethat steps can be executed in different sequence where this is logicallypossible.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a support” includes a plurality of supports. In thisspecification and in the claims that follow, reference will be made to anumber of terms that shall be defined to have the following meaningsunless a contrary intention is apparent.

DEFINITIONS

In describing and claiming the disclosed subject matter, the followingterminology will be used in accordance with the definitions set forthbelow.

By “administration” is meant introducing a compound, biologicalmaterials including a cell population, or a combination thereof, of thepresent disclosure into a human or animal subject. The preferred routeof administration of the compounds is intravenous. However, any route ofadministration, such as oral, topical, subcutaneous, peritoneal,intraarterial, inhalation, vaginal, rectal, nasal, introduction into thecerebrospinal fluid, or instillation into body compartments can be used.Direct injection into a target tissue site such as a solid tumor is alsocontemplated.

The terms “therapeutic agent”, “chemotherapeutic agent”, or “drug” asused herein refers to a compound or a derivative thereof that caninteract with a cancer cell, thereby reducing the proliferative statusof the cell and/or killing the cell. Examples of chemotherapeutic agentsinclude, but are not limited to, alkylating agents (e.g.,cyclophosphamide, ifosamide), metabolic antagonists (e.g., methotrexate(MTX), 5-fluorouracil or derivatives thereof), antitumor antibiotics(e.g., mitomycin, adriamycin), plant-derived antitumor agents (e.g.,vincristine, vindesine, Taxol), cisplatin, carboplatin, etoposide, andthe like. Such agents may further include, but are not limited to, theanti-cancer agents TRIMETHOTRIXATE™ (TMTX), TEMOZOLOMIDE™,RALTRITREXED™, S-(4-Nitrobenzyl)-6-thioinosine (NBMPR),6-benzyguanidine(6-BG), bis-chloronitrosourea (BCNU) and CAMPTOTHECIN™, or a therapeuticderivative of any thereof.

The term “therapeutically effective amount” as used herein refers tothat amount of the compound being administered that will relieve to someextent one or more of the symptoms of a disease, a condition, or adisorder being treated. In reference to cancer or pathologies related tounregulated cell division, a therapeutically effective amount refers tothat amount which has the effect of (1) reducing the size of a tumor,(2) inhibiting (that is, slowing to some extent, preferably stopping)aberrant cell division, for example cancer cell division, (3) preventingor reducing the metastasis of cancer cells, and/or, (4) relieving tosome extent (or, preferably, eliminating) one or more symptomsassociated with a pathology related to or caused in part by unregulatedor aberrant cellular division, including for example, cancer, orangiogenesis.

The terms “treating” or “treatment” of a disease (or a condition or adisorder) as used herein refer to preventing the disease from occurringin an animal that may be predisposed to the disease but does not yetexperience or exhibit symptoms of the disease (prophylactic treatment),inhibiting the disease (slowing or arresting its development), providingrelief from the symptoms or side-effects of the disease (includingpalliative treatment), and relieving the disease (causing regression ofthe disease). With regard to cancer, these terms also mean that the lifeexpectancy of an individual affected with a cancer may be increased orthat one or more of the symptoms of the disease will be reduced.

The terms “subject” and “patient” as used herein include humans, mammals(e.g., cats, dogs, horses, etc.), living cells, and other livingorganisms. A living organism can be as simple as, for example, a singleeukaryotic cell or as complex as a mammal. Typical hosts to whichembodiments of the present disclosure may be administered will bemammals, particularly primates, especially humans. For veterinaryapplications, a wide variety of subjects will be suitable, e.g.,livestock such as cattle, sheep, goats, cows, swine, and the like;poultry such as chickens, ducks, geese, turkeys, and the like; anddomesticated animals particularly pets such as dogs and cats. Fordiagnostic or research applications, a wide variety of mammals will besuitable subjects, including rodents (e.g., mice, rats, hamsters),rabbits, primates, and swine such as inbred pigs and the like. In someembodiments, a system includes a sample and a subject. The term “livinghost” refers to host or organisms noted above that are alive and are notdead. The term “living host” refers to the entire host or organism andnot just a part excised (e.g., a liver or other organ) from the livinghost.

The term “γδ T-cells (gamma delta T-cells)” as used herein refers to asmall subset of T-cells that can specifically bind to a distinct T-cellreceptor (TCR) on their surface. A majority of T-cells have a TCRcomposed of two glycoprotein chains called α- and β-TCR chains. Incontrast, in γδ T-cells, the TCR is made up of one γ-chain and oneδ-chain. This group of T-cells is usually much less common than αβT-cells, but are found at their highest abundance in the gut mucosa,within a population of lymphocytes known as intraepithelial lymphocytes(IELs).

The antigenic molecules that activate γδ T-cells are still largelyunknown. However, γδ T-cells are peculiar in that they do not seem torequire antigen processing and MHC presentation of peptide epitopesalthough some recognize MHC class IB molecules. Furthermore, γδ T-cellsare believed to have a prominent role in recognition of lipid antigens,and to respond to stress-related antigens such as, MIC-A and MIC-B.

The term “cancer”, as used herein, shall be given its ordinary meaning,as a general term for diseases in which abnormal cells divide withoutcontrol. In particular, and in the context of the embodiments of thepresent disclosure, cancer refers to angiogenesis-related cancer. Cancercells can invade nearby tissues and can spread through the bloodstreamand lymphatic system to other parts of the body. There are several maintypes of cancer, for example, carcinoma is cancer that begins in theskin or in tissues that line or cover internal organs. Sarcoma is cancerthat begins in bone, cartilage, fat, muscle, blood vessels, or otherconnective or supportive tissue. Leukemia is cancer that starts inblood-forming tissue such as the bone marrow, and causes large numbersof abnormal blood cells to be produced and enter the bloodstream.Lymphoma is cancer that begins in the cells of the immune system.

When normal cells lose their ability to behave as a specified,controlled and coordinated unit, a tumor is formed. Generally, a solidtumor is an abnormal mass of tissue that usually does not contain cystsor liquid areas (some brain tumors do have cysts and central necroticareas filled with liquid). A single tumor may even have differentpopulations of cells within it, with differing processes that have goneawry. Solid tumors may be benign (not cancerous), or malignant(cancerous). Different types of solid tumors are named for the type ofcells that form them. Examples of solid tumors are sarcomas, carcinomas,and lymphomas. Leukemias (cancers of the blood) generally do not formsolid tumors.

Representative cancers include, but are not limited to, bladder cancer,breast cancer, colorectal cancer, endometrial cancer, head and neckcancer, leukemia, lung cancer, lymphoma, melanoma, non-small-cell lungcancer, ovarian cancer, prostate cancer, testicular cancer, uterinecancer, cervical cancer, thyroid cancer, gastric cancer, brain stemglioma, cerebellar astrocytoma, cerebral astrocytoma, glioblastoma,ependymoma, Ewing's sarcoma family of tumors, germ cell tumor,extracranial cancer, Hodgkin's disease, leukemia, acute lymphoblasticleukemia, acute myeloid leukemia, liver cancer, medulloblastoma,neuroblastoma, brain tumors generally, non-Hodgkin's lymphoma,osteosarcoma, malignant fibrous histiocytoma of bone, retinoblastoma,rhabdomyosarcoma, soft tissue sarcomas generally, supratentorialprimitive neuroectodermal and pineal tumors, visual pathway andhypothalamic glioma, Wilms' tumor, acute lymphocytic leukemia, adultacute myeloid leukemia, adult non-Hodgkin's lymphoma, chroniclymphocytic leukemia, chronic myeloid leukemia, esophageal cancer, hairycell leukemia, kidney cancer, multiple myeloma, oral cancer, pancreaticcancer, primary central nervous system lymphoma, skin cancer, small-celllung cancer, among others.

A tumor can be classified as malignant or benign. In both cases, thereis an abnormal aggregation and proliferation of cells. In the case of amalignant tumor, these cells behave more aggressively, acquiringproperties of increased invasiveness. Ultimately, the tumor cells mayeven gain the ability to break away from the microscopic environment inwhich they originated, spread to another area of the body (with a verydifferent environment, not normally conducive to their growth), andcontinue their rapid growth and division in this new location. This iscalled metastasis. Once malignant cells have metastasized, achieving acure is more difficult. Benign tumors have less of a tendency to invadeand are less likely to metastasize.

Brain tumors spread extensively within the brain but do not usuallymetastasize outside the brain. Gliomas are very invasive inside thebrain, even crossing hemispheres. They do divide in an uncontrolledmanner, though. Depending on their location, they can be just as lifethreatening as malignant lesions. An example of this would be a benigntumor in the brain, which can grow and occupy space within the skull,leading to increased pressure on the brain.

The term “reducing a cancer” as used herein refers to a reduction in thesize or volume of a tumor mass, a decrease in the number of metastasizedtumors in a subject, a decrease in the proliferative status (the degreeto which the cancer cells are multiplying) of the cancer cells, and thelike.

The terms “isolated’ and isolated population of cells” as used hereinrefers to a cell or a plurality of cells removed from the tissue orstate in which they are found in a subject. The terms may furtherinclude cells that have been separated according to such parameters as,but not limited to, cell surface markers, a reporter marker such as adye or label,

The term “expressed” or “expression” as used herein refers to thetranscription from a gene to give an RNA nucleic acid molecule at leastcomplementary in part to a region of one of the two nucleic acid strandsof the gene. The term “expressed” or “expression” as used herein alsorefers to the translation from said RNA nucleic acid molecule to give aprotein, a polypeptide, or a portion or fragment thereof.

The term “promoter” as used herein refers to the DNA sequence thatdetermines the site of transcription initiation from an RNA polymerase.A “promoter-proximal element” may be a regulatory sequence within about200 base pairs of the transcription start site.

The term “recombinant cell” refers to a cell that has a new combinationof nucleic acid segments that are not covalently linked to each other innature. A new combination of nucleic acid segments can be introducedinto an organism using a wide array of nucleic acid manipulationtechniques available to those skilled in the art. A recombinant cell canbe a single eukaryotic cell, or a single prokaryotic cell, or amammalian cell. The recombinant cell may harbor a vector that isextragenomic. An extragenomic nucleic acid vector does not insert intothe cell's genome. A recombinant cell may further harbor a vector or aportion thereof that is intragenomic. The term “intragenomic” defines anucleic acid construct incorporated within the recombinant cell'sgenome.

The terms “recombinant nucleic acid” and “recombinant DNA” as usedherein refer to combinations of at least two nucleic acid sequences thatare not naturally found in a eukaryotic or prokaryotic cell. The nucleicacid sequences include, but are not limited to, nucleic acid vectors,gene expression regulatory elements, origins of replication, suitablegene sequences that when expressed confer antibiotic resistance,protein-encoding sequences, and the like. The term “recombinantpolypeptide” is meant to include a polypeptide produced by recombinantDNA techniques such that it is distinct from a naturally occurringpolypeptide either in its location, purity or structure. Generally, sucha recombinant polypeptide will be present in a cell in an amountdifferent from that normally observed in nature.

The terms “operably” or “operatively linked” as used herein refer to theconfiguration of the coding and control sequences so as to perform thedesired function. Thus, control sequences operably linked to a codingsequence are capable of effecting the expression of the coding sequence.A coding sequence is operably linked to or under the control oftranscriptional regulatory regions in a cell when DNA polymerase willbind the promoter sequence and transcribe the coding sequence into mRNAthat can be translated into the encoded protein. The control sequencesneed not be contiguous with the coding sequence, so long as theyfunction to direct the expression thereof. Thus, for example,intervening untranslated yet transcribed sequences can be presentbetween a promoter sequence and the coding sequence and the promotersequence can still be considered “operably linked” to the codingsequence.

The terms “heterologous” and “exogenous” as they relate to nucleic acidsequences such as coding sequences and control sequences, denotesequences that are not normally associated with a region of arecombinant construct or with a particular chromosomal locus, and/or arenot normally associated with a particular cell. Thus, a “heterologous”region of a nucleic acid construct is an identifiable segment of nucleicacid within or attached to another nucleic acid molecule that is notfound in association with the other molecule in nature. For example, aheterologous region of a construct tip could include a coding sequenceflanked by sequences not found in association with the coding sequencein nature. Another example of a heterologous coding sequence is aconstruct where the coding sequence itself is not found in nature (e.g.,synthetic sequences having codons different from the native gene).Similarly, a host cell transformed with a construct which is notnormally present in the host cell would be considered heterologous forpurposes of this invention.

In some embodiments the promoter will be modified by the addition ordeletion of sequences, or replaced with alternative sequences, includingnatural and synthetic sequences as well as sequences which may be acombination of synthetic and natural sequences. Many eukaryoticpromoters contain two types of recognition sequences: the TATA box andthe upstream promoter elements. The former, located upstream of thetranscription initiation site, is involved in directing RNA polymeraseto initiate transcription at the correct site, while the latter appearsto determine the rate of transcription and is upstream of the TATA box.Enhancer elements can also stimulate transcription from linkedpromoters, but many function exclusively in a particular cell type. Manyenhancer/promoter elements derived from viruses, e.g. the SV40, the Roussarcoma virus (RSV), and CMV promoters are active in a wide array ofcell types, and are termed “constitutive” or “ubiquitous.” The nucleicacid sequence inserted in the cloning site may have any open readingframe encoding a polypeptide of interest, with the proviso that wherethe coding sequence encodes a polypeptide of interest, it should lackcryptic splice sites which can block production of appropriate mRNAmolecules and/or produce aberrantly spliced or abnormal mRNA molecules.

The termination region which is employed primarily will be one ofconvenience, since termination regions appear to be relativelyinterchangeable. The termination region may be native to the intendednucleic acid sequence of interest, or may be derived from anothersource.

The term “vector” as used herein refers to a polynucleotide comprised ofsingle strand, double strand, circular, or supercoiled DNA or RNA. Atypical vector may be comprised of the following elements operativelylinked at appropriate distances for allowing functional gene expression:replication origin, promoter, enhancer, 5′ mRNA leader sequence,ribosomal binding site, nucleic acid cassette, termination andpolyadenylation sites, and selectable marker sequences. One or more ofthese elements may be omitted in specific applications. The nucleic acidcassette can include a restriction site for insertion of the nucleicacid sequence to be expressed. In a functional vector the nucleic acidcassette contains the nucleic acid sequence to be expressed includingtranslation initiation and termination sites.

A vector is constructed so that the particular coding sequence islocated in the vector with the appropriate regulatory sequences, thepositioning and orientation of the coding sequence with respect to thecontrol sequences being such that the coding sequence is transcribedunder the “control” of the control or regulatory sequences. Modificationof the sequences encoding the particular protein of interest may bedesirable to achieve this end. For example, in some cases it may benecessary to modify the sequence so that it may be attached to thecontrol sequences with the appropriate orientation; or to maintain thereading frame. The control sequences and other regulatory sequences maybe ligated to the coding sequence prior to insertion into a vector.Alternatively, the coding sequence can be cloned directly into anexpression vector which already contains the control sequences and anappropriate restriction site which is in reading frame with and underregulatory control of the control sequences.

The term “lentiviral-based vector” as used herein refers to a lentiviralvector designed to operably insert an exogenous polynucleotide sequenceinto a host genome in a site-specific manner. Lentiviral-based targetingvectors may be based on, but is not limited to, for example, HIV-1,HIV-2, simian immunodeficiency virus (SIV), or feline immunodeficiencyvirus (Fly). In a preferred embodiment, the lentiviral-based targetingvector is an HIV-based targeting vector. This vector may comprise all ora portion of the polynucleotide sequence of HIV.

The terms “transformation”, “transduction” and “transduction” all denotethe introduction of a polynucleotide into a recipient cell or cells.

Discussion

A major limitation to chemotherapy treatments for cancer is drug inducedimmune toxicity. This results, upon administration of the therapeuticagent, in the killing of immunocompetent cells and loss of an effectiveimmune system that would otherwise ward off undesirable infections orprovide a defense against cancer cells. One strategy to combat thesevere toxic effects of chemotherapy is to genetically engineer blood ormarrow cells by the introduction of retroviral vectors designed toexpress cDNA sequences that confer drug resistance. The introduction ofdrug resistant genes into hematopoietic stem cells (HSCs) results intransgene expression throughout the entire host hematopoietic system,including immunocompetent cells such as T cells and natural killercells, after transplantation of gene-modified cells back into arecipient patient, as described by McMillin et al., (2006) Human GeneTherapy 17:798-806. The patient can then develop an active immune systemwhile at the same time undergoing chemotherapy. However, in the case ofHSC transgene expression, over time not all of the T cells and naturalkiller cells in the subject express the drug resistant gene. Forexample, McMillin et al. (2005) discloses that less than 50% of NK-cellscontained an expressing marker 8 week post-transplant. See FIG. 3 ofMcMillin et al.

An alternative strategy would be to selectively genetically modifycytotoxic immunocompetent cells that can actively target those cancercells able to resist the simultaneous administration of achemotherapeutic agent, thereby effectively eliminating most if not allcancer cells from the patient.

In certain embodiments, the invention relates to isolated compositionscomprising natural killer cells wherein greater than about 50% of thenatural killer cells express a polypeptide that confers resistance to achemotherapy agent. In other embodiments, the invention relates toisolated compositions comprising natural killer T-cells wherein greaterthan about 50% of the natural killer T-cells comprise a nucleic acidthat encodes a polypeptide that confers resistance to a chemotherapyagent. In other embodiment the invention relates to isolatedcompositions consisting essentially of natural killer T-cells comprisinga nucleic acid that encodes a polypeptide that confers resistance to achemotherapy agent. In certain embodiments the polypeptide that confersresistance to a chemotherapy agent is O⁶ methylguanine DNAmethyltransferase (MGMT), a drug resistant variant of dihydrofolatereductase (L22Y-DHFR), thymidylate synthase, multiple drug resistance-1protein (MDR1).

In certain embodiments, the invention relates to methods of treating asubject diagnosed with cancer comprising: administering a chemotherapyagent to the subject and administering a chemotherapy resistant naturalkiller cell composition to the subject wherein the chemotherapyresistant natural killer cell composition comprises natural killer cellsgenetically engineering to express a polypeptide that confers resistanceto the chemotherapy agent.

The present disclosure encompasses methods whereby immunocompetent cellsare selectively protected from the toxic effects of chemotherapy,thereby allowing co-administration of chemotherapy and cell-basedimmunotherapy, and hence termed drug resistant immunotherapy. Thefeasibility of using drug resistant immunotherapy in the context ofdrug-resistant hematopoietic cells has been shown (Cesano et al., (1998)Anticancer Res. 18: 2289-2295). Mouse bone marrow cells were geneticallyengineered by retroviral-mediated introduction of a cDNA encoding for amutant form of DHFR, i.e. L22Y-DHFR that confers resistance totrimetrexate (TMTX). Mice were transplanted with gene-modified bonemarrow cells that resulted in transgene expression in all hematopoieticlineages. The mice were then treated with the immunotherapeutic agentanti-CD 137, TMTX alone, or a combination of anti-CD 137 and TMTX.

In mice inoculated with AG104 sarcoma cells, TMTX chemotherapy reducedthe efficacy of an anti-CD137 antibody in mice transplanted withnon-modified cells that were sensitive to the TMTX. However, when micewere protected against chemotherapy-induced toxicity throughtransplantation of L22Y-DHFR-expressing bone marrow, the combinedtreatment of TMTX and anti-CD137 resulted in complete eradication oftumors in 100% of animals. The present disclosure provides evidence thatgenetically engineered human immunocompetent cells can be used in thecontext of drug resistance immunotherapy, rather than geneticallymodifying the entire hematopoietic system. The ability to provide to apatient a genetically-modified population of cytotoxic immunocompetentcells, and particular if delivered locally to the site of a tumor, wouldallow for immunotherapy in conjunction with chemotherapy withoutnecessarily undergoing bone marrow transplantation.

NK-92 and TALL-104 cells are representative immune-effector cell linessince both of these cell types recognize and kill a wide range ofmalignant cells, including K562 cells (Sawai et al., (2001) Mol. Ther.3: 78-87; Tam et al., (1999) J. Hematother. 8: 281-290). The highlypotent cytotoxic human NK cell line NK-92 is an interleukin-2(IL-2)-dependent human natural killer cell line with functional andphenotypic characteristics of activated NK cells (Gong et al., (1994)Leukemia 8: 652-658). NK-92 cells are effectors of the innate immunesystem, which play an important role in host responses against virusesand tumor cells Due to the high cytotoxicity against a broad spectrum ofprimary and established tumor cells at low effector:target ratios andagainst primary leukemia in SCID mice (Gong et al., (1994) Leukemia 8:652-658; Yan et al., (1998) Clin. Cancer Res. 4: 2859-2868; Tam et al.,(1999) J. Hematother. 8: 281-290) makes them a reasonable candidate as adrug resistant immune effector cell (Yan et al., (1998) Clin. CancerRes. 4: 2859-2868; Tam et al., (1999) J. Hematother. 8: 281-290).TALL-104 cells are an interleukin 2-dependent leukemic T cell line thathas surface markers typical of those found on both cytotoxic Tlymphocytes and natural killer cells. TALL-104 cells lyze tumor cells ina non-HLA-restricted fashion (Tam et al., (1999) J. Hematother. 8:281-290). Adoptive immunotherapy with TALL-104 has induced long-termcomplete or partial remissions in tumor bearing animals (Tam et al.,(1999) J. Hematother. 8: 281-290; Geoerger et al., (2000) Neuro Oncol.2: 103-113). Similar to NK-92 cells, we used TALL-104 cells asimmunocompetent cells for our proof-of-concept studies since these cellscan be expanded in culture indefinitely to provide an unlimited sourceof effector cells with stable tumoricidal activity.

P140K-MGMT-genetically engineered NK-92 and TALL-104 cells wereresistant to TMZ and had cytotoxic activities similar to thenon-modified cells. Additionally, the gene-modified cells showedcytolytic activities similar to non-transduced cells after drugselection. Therefore, genetic modification of these cells does notaffect their cytotoxic activity.

Drug resistant immunotherapy was evaluated in a series of cytotoxicassays, in the presence and absence of a cytotoxic drug. Dasqupta etal., (2010) Biochem. Biophys. Res. Comm. 391:170-175. Importantly,gene-modified immunocompetent cells displayed significant cytolyticactivities toward drug resistant tumor cells in the presence of drug. Incontrast, non-modified immunocompetent cells were ineffective at tumorkilling when drug was administered. Combined, these results demonstratethat in the presence of a cytotoxic chemotherapeutic drug, gene-modifiedeffector cells remain active, and a greater level of target cancer cellkilling was observed after treating gene-modified effector cells andnon-modified target cells compared to non-modified effector cells anddrug resistant target cells. Accordingly, genetically-modified drugresistant immunocompetent cells could be engineered to survive thecytotoxic effects of chemotherapeutic agents and the effectiveness oftumor killing significantly increases during a chemotherapy challenge.

The present disclosure provides data that drug-resistant immunocompetenteffector cells are superior cytotoxic effectors during a chemotherapychallenge. This is a significant finding which can be combined withcurrent cell-based and adoptive immunotherapies. It has been shown thatregression of large, vascularized tumors occurs in patients withrefractory metastatic melanoma. However, for maximum effectiveness, alympho-depleting regimen is typically necessary prior to autologouslymphocyte cell transfer (Chinnasamy et al., (2004) Hum. Gene Ther. 8:758-769).

The generation and expansion of drug-resistant lymphocytes (as opposedto the entire hematopoietic system) ex vivo can allow, in this setting,for administration of immunocompetent cell-based therapy concurrentlywith chemotherapy, potentially improving tumor clearance while antitumorimmunity is established and maintained. In this scenario, non-transducedlymphocytes can be depleted using a selective chemotherapy treatment,which could be continually applied during the administration of adoptiveimmunotherapy. The co-administration of chemo- and immunotherapies wouldthen lead to long-term tumor clearance.

It was shown, however, that the growth of CML cells in mice transplantedwith bone marrow engineered to confer resistance to MTX can beexacerbated by the administration of chemotherapy (Rosenberg & Dudley(2004) Proc. Natl. Acad. Sci. U S A. 101: 14639-14645). Thuschemotherapy treatment in the context of gene-modified whole bone marrowprotection may induce secondary effects such as immune suppression thatallow some cancers to survive a drug challenge. Base on the results ofthe present disclosure, however, instead of transplanting drug resistanthematopoietic stem cells, a more effective strategy involvestransplantation of drug resistant immunocompetent lymphocytes.

Additionally, it was recently shown that melanoma and glioma cell linesare sensitive to the combination of TMZ and antifolates (Sweeney et al.,(2002) J. Pharmacol. Exp. Ther. 300: 1075-1084). In one embodiment ofthe methods of the disclosure, therefore, retroviral transfer of dualvectors that co-express drug resistant variants of DHFR, such as,L22Y-DHFR, together with P140K-MGMT would increase tumor cell killing byallowing effective cytotoxic immunotherapy while administering acombination of chemotherapeutic agents. Expression of DHFR mutants, forexample, can provide resistance to antifolates such as methotrexate andtrimetrexate, while MGMT expression can provide resistance tomonofunctional methylating agents such as dacarbazine and procarbazineas well as bifunctional chloroethylating agents such as BCNU, ACNU orTMZ.

Accordingly, a series of combinatorial cytotoxicity assays was performedwith non-modified and gene-modified effector and target cells. Todetermine the effects of TMZ on non-modified cells, cytotoxicity assayswere performed whereby non-modified effector cells were mixed withgene-modified target cells in either the absence or presence of 200 μM6-BG/TMZ.

Gene-modified target cells were used to eliminate the effects ofchemotherapy on the target cells (gene-modified target cells wereresistant to TMZ at this drug concentration). Before these studies wereinitiated, the sensitivity of gene-modified K562 cells to NK92 andTa11-104 cells was determined. A 4 hr cytotoxicity assay was conductedwhere non-modified effector cells (E) were incubated with eithernon-modified target (T) or gene-modified target (Tm) cells at aneffector to target ratio of 10:1, as shown in FIG. 27, in the absence ofdrug. The cytotoxicities of both the non-modified effector cell lines(NK92 and TALL-104) toward either non-modified or gene-modified targetcells were comparable (P_(NK-92)=0.8441, P_(TALL-014)=0.6349). Thusgenetic modification of the target cells did not affected their lyses bythe immunocompetent cells.

Cytotoxic assays were then conducted using non-modified effector cellsand gene-modified target cells in the presence of TMZ. A significantdecrease in both NK-92 and TALL-104 cell mediated lysis was observedwhen compared to gene-modified target cells in the absence of drugtreatment (see FIG. 27; P_(NK-92)=0.0003, P_(TALL-014)=0.0008). Thus,the clearance of drug resistant tumor cells by non-modifiedimmunocompetent cells is severely limited after a chemotherapychallenge.

To compare the killing effectiveness of non-modified and gene-modifiedimmune effector cells during drug treatments, cytotoxicity assays wereconducted whereby non-modified or P140KMGMT-modified effector cells (Em)were incubated with gene-modified target cells (Tm) and 200 μM 6-BG/TMZ.

When compared to non-modified effector cells, genetically-modified NK-92cells lyzed target cells significantly better after being treated with6-BG/TMZ, as shown in FIG. 28A (P_(NK-92)=0.0001). Thus, in the presenceof drug, P140KMGMT-modified immunocompetent cells were active in killingtumor cells. Under identical conditions, however, genetically engineeredTALL-104 cells had only a modest increase in cytotoxic activity (FIG.28B).

To determine the effectiveness of drug resistant tumor cells during achemotherapy challenge when the target cells are sensitive to the drugtreatment, gene-modified effectors, i.e. P140KMGMT-NK-92 andP140KMGMT-TALL-104 cells, were incubated with non-modified,drug-sensitive target cells and 200 μM 6-BG/TMZ. The cytotoxicities ofthese drug resistant immunocompetent cells were then compared with thecytotoxicities achieved using drug-sensitive immunocompetent cells, asshown in FIGS. 28C and 28D.

Compared to the killing of gene-modified target cells by non-modifiedeffector cells, there was a significant increase of about 4.5-fold and2.5-fold killing of non-modified target cells by the geneticallymodified NK-92 and TALL-104 cells, respectively (P_(NK-92)=0.0012,P_(TALL-014)=0.0011). These data demonstrate that P140KMGMT-modifiedNK-92 and TALL-104 cells function as potent effectors in the presence6-BG/TMZ, and that the drug resistant immunocompetent cells, when usedconcurrently with chemotherapy, can significantly enhance the killing oftarget cells.

Embodiments of the present disclosure encompass methods of treatingcancers, and in particular cancerous tumors. The methods of thedisclosure combine the use of chemotherapeutic agents that can kill orreduce the proliferation of cancerous cells, with immunotherapy toeffectively eliminate those cancerous cells that develop drug resistanceor otherwise escape the chemotherapeutic agent. The methods of thepresent disclosure provide for isolating cytotoxic immune cells,including, but not limited to, γδ T-cells either from a patient to betreated or from another source, as described, for example, by Lamb L. S.in U.S. Pat. No. 7,078,034, incorporated herein by reference in itsentirety. The isolated cells may then be transfected with a nucleic acidvector comprising a heterologous nucleotide sequence encoding apolypeptide that confers resistance to a selected chemotherapeutic agentto the cell. The patient in need of treatment for a cancer, and inparticular a tumor, may then receive a dose, or doses, of thetransfected T-cells before, after or with the chemotherapeutic agent.The agent itself, while intended to be toxic to the targeted cancercells, and will reduce the proliferation and viability of the cells, mayalso induce the formation on the cell surface of the cancer cells ofstress-related proteins. Transfected γδ T-cells, for example, have thecharacteristic of being able to recognize and therefore target suchstress-related ligands, thereby specifically or preferentially targetingthe cancer cells.

The transduction of a population of cytotoxic immune cells, such as γδT-cells with a heterologous nucleic acid encoding an exogenouspolypeptide conferring resistance to the chemotherapeutic agent canensure that the immunotherapeutic cells are not adversely affected bythe agent (drug). The result is that the chemotherapy and theimmunotherapy cooperate to efficiently reduce the tumor mass oreliminate the cancerous cells. The data provided herein indicate that anincrease in the survival outcome of a treated animal can be achieved.

It is contemplated that the genetically modified cytotoxic immune cellssuch as γδ T-cells may be delivered to the targeted tumor directly bysuch as, but not limited to, direct injection into the tumor mass,delivery to a blood vessel entering the tumor mass, or a combination ofboth. For example, it is contemplated that the cells may be delivered toa glioblastomal mass in the brain of a patient by direct implantationthrough a cannulated needle inserted into the tumor mass.

The methods of the disclosure compare with other methods that combinechemotherapeutic and immunologic approaches to treating cancers. Forexample, as shown in FIG. 1, cytokines and other factors that canstimulate the immune system, including the innate system, may beadministered to a patient systemically, resulting in expansion of manyclasses of cells of the patients entire immune system. However, when thechemotherapeutic agent is then administered, the toxicity of the agentcan effectively reduce or destroy the immune system cells themselves,thereby eliminating the potential benefits of an expanded immune system.

An alternative protocol, as shown in FIG. 17, comprises isolating bonemarrow cells from a subject and transducing the cells with a nucleicacid vector, such as, but not limited to, a lentiviral vector, where thevector comprises a heterologous nucleic acid sequence encoding apolypeptide that can confer resistance to the chemotherapeutic agentselected for use in treating a cancer, as shown in FIG. 13, for example.In an experimental system, as shown in FIG. 17, the transduced marrowcells may be transplanted into a recipient subject that has had theimmune system destroyed by high-level radiation. If these subjects thenreceive a tumor cell inoculation they will develop a tumor(s), as shownin FIGS. 18 and 19, and discussed in McMillin et al., (2006) Hum. GeneTherapy 17: 798-806, incorporated herein by reference in its entirety.If the subject then receives either the selected chemotherapeutic agentor an inducer of cancer-targeting immune cells (in this case byadministering anti-CD137 antibodies), then reductions in the sizes oftumors (in FIGS. 18 and 19, AG104 sarcoma tumors) are observed.

In some protocols that combine immunotherapy and chemotherapy to treatcancers in a subject human or animal patient, cytokines (IL-2, IL-12,GM-CSF, and the like) may be delivered to a patient to boost theformation of cytotoxic lymphocytes. In other methods, an anti-CD137specific antibody may be employed. CD137 is a member of the tumornecrosis factor (TNF)/nerve growth factor (NGF) family of receptors andis expressed by activated T- and B-lymphocytes and monocytes; its ligandhas been found to play an important role in the regulation of immuneresponses. An anti-CD137 monoclonal antibody can specifically bind toCD137-expressing immune cells such as activated T-cells and freshlyisolated mouse dendritic cells (DCs), thereby stimulating an immuneresponse, in particular of a cytotoxic T cell response, against tumorcells.

It has also been observed that the reduction in tumors may be transientwhen a chemotherapeutic agent is used (FIG. 18), and theimmunotherapeutic reduction in tumor size may also show a rebound (FIG.19). In contrast, if the chemotherapeutic agent and the immunotherapyare administered together or sequentially to the subject, then asignificant and prolonged reduction in tumor size is seen (FIG. 20).Survival of the subject treated animal is also increased. Transfer ofsplenocytes from such a successfully treated subject to a subjectinjected with a cancer cell population resulted in increased survival(FIG. 21) showing the prolongation of cancer-specific cells after curingof a cancer. The experiments, as summarized in FIG. 22, show thatapplication of transduction of a drug-resistance to immune system cellsallows for the practical application of a combination of chemotherapyand immunotherapy to increase survival, and destruction of tumors. Thismethod has also been applied to the regression of very large tumors, asshown in FIG. 23.

While treatment protocols for use against cancerous tumors has been ofsome success, as evidenced by the data presented in FIGS. 15-23, thesuccess of such methods with glioblastomal tumors has been minimal, withprolonged survival of the patient not extending beyond about 24 months.Accordingly, the methods of the present disclosure provide analternative immunotherapy step that employsisolated cytotoxic immunecells, and particularly the subpopulation of γδ T-cells that canspecifically recognize, bind to, and destroy cancer cells that producethe cell-surface stress antigen MICA/B. γδ T-cells comprise only about5% of the total circulating T-cells and form a powerful component of theinnate defense system. In the methods of the disclosure, CD4-CD8-cellsmay be isolated from T-cell populations by such well known methods asFACS and cultured in vitro to expand the population size.

Accordingly, the methods of the present disclosure provide cytotoxicimmune cells, such as γδ T-cells, that are genetically modified tocomprise a heterologous nucleic acid that, when expressed in the cellsconfers upon them resistance to the chemotherapeutic agent. The modifiedT-cells are then able to survive for sufficient time to effectivelydestroy most if not all of the target cancer cells.

It has now been shown that animals engrafted with glioblastomal cellshave a significantly increased survival time when provided the combinedtreatment of a chemotherapeutic agent and the appropriate geneticallymodified agent-resistant γδ T-cells that are resistant to thechemotherapeutic agent, compared to animals that have received only theagent.

Genetic modification of the isolated cytotoxic immune cells may be byany method known in the art. For example, but not intended to belimiting, isolated γδ T-cells may be transfected with a lentiviralvector such as SIV comprising a heterologous nucleic acid sequenceencoding a variant of the protein MGMT (e.g. a P104K variant).Efficiency of transduction may be shown by co-transfecting the cellswith a lentivirus vector comprising a nucleic acid sequence encoding areporter protein such as, but not limited to, enhanced green fluorescentprotein (EGFP) or the like. Transfer of MGMT to cells confers on themresistance to DNA alkylating agents, as shown, for example, in FIG. 7.Similarly, using a recombinant SIV lentivirus vector, γδ T-cells may betransfected with such as MGMT, as shown in FIG. 8.

One aspect of the present disclosure, therefore, encompasses methods forreducing a cancer in a patient, comprising the steps of: obtaining apopulation of isolated cytotoxic immune cells, where the isolatedcytotoxic immune cells have been genetically modified to be resistant toa therapeutic agent; administering to a patient in need thereof, aneffective amount of the therapeutic agent; and administering to thepatient population of isolated genetically modified cytotoxic immunecells, whereupon the cytotoxic immune cells are delivered to the tumor,thereby reducing the cancer in the patient.

In embodiments of this aspect of the disclosure, the isolated cytotoxicimmune cells can be γδ T-cells.

In embodiments of this aspect of the disclosure, the isolated cytotoxicimmune cells can be isolated from the patient having the cancer.

In some embodiments of this aspect of the disclosure, the isolatedcytotoxic immune cells may be isolated from a source other than thepatient in need thereof.

In embodiments of this aspect of the disclosure, the therapeutic agentcan have the characteristic of inducing a stress protein in a cancercell of the patient, where the stress protein is recognized by thecytotoxic immune cells.

In embodiments of this aspect of the disclosure, the therapeutic agentcan be a cytotoxic chemotherapeutic agent characterized by a celldeveloping resistance to said therapeutic agent when the cell receives aheterologous nucleic acid, and wherein the heterologous nucleic acid isexpressed in the cell.

In the embodiments of this aspect of the disclosure, the therapeuticagent can be a cytotoxic chemotherapeutic agent selected from the groupconsisting of: an alkylating agent, a metabolic antagonist, an antitumorantibiotic, and a plant-derived antitumor agent.

In some embodiments of this aspect of the disclosure, the therapeuticagent can be a cytotoxic chemotherapeutic agent selected from the groupconsisting of: a cyclophosphamide, an ifosamide, a methotrexate, asubstituted nucleotide, a substituted nucleoside, fluorouracil, amitomycin, adriamycin, vincristine, vindesine, Taxol, cisplatin,carboplatin, and etoposide.

In embodiments of this aspect of the disclosure, the therapeutic agentcan be selected from the group consisting of: TRIMETHOTRIXATE™ (TMTX),methotrixate (MTX), TEMOZOLOMIDE™, RALTRITREXED™,S-(4-Nitrobenzyl)-6-thioinosine (NBMPR), CAMPTOTHECIN™,6-benzylguanidine, and a therapeutic derivative of any thereof.

In embodiments of this aspect of the disclosure, the step of obtaining apopulation of isolated cytotoxic immune cells genetically modified to beresistant to a therapeutic agent can comprise: isolating from a subjecthuman or animal a population of cytotoxic immune cells; culturing theisolated population of cytotoxic immune cells, thereby increasing thepopulation of the cells; stably transfecting the population of cytotoxicimmune cells with a vector comprising a heterologous nucleic acidsequence operably linked to a promoter, wherein the heterologous nucleicacid sequence encodes a polypeptide conferring to the cell resistance tothe therapeutic agent.

In embodiments of this aspect of the disclosure, the population ofstably transfected cytotoxic immune cells can be viably maintained.

In some embodiments of this aspect of the disclosure, the therapeuticagent can be TRIMETHOTRIXATE™ or methotrexate, and the heterologousnucleic acid sequence encodes dihydrofolate reductase, or a derivativethereof.

In other embodiments of this aspect of the disclosure, the therapeuticagent can be TEMOZOLOMIDE™, or a therapeutically agent derivativethereof, and the heterologous nucleic acid sequence may encode O⁶methylguanine DNA methyltransferase, or a derivative thereof.

In embodiments of this aspect of the disclosure, the isolatedgenetically modified cytotoxic immune cells and the therapeutic agentcan be co-administered to the patient.

In some embodiments of this aspect of the disclosure, the geneticallymodified cytotoxic immune cells and the therapeutic agent can besequentially administered to the patient.

In embodiments of this aspect of the disclosure, the geneticallymodified cytotoxic immune cells are administered to the patient directlyinto the tumor or to a blood vessel proximal and leading into the tumor.

In some embodiments of this aspect of the disclosure, the tumor is aglioblastoma.

Yet another aspect of the present disclosure provides systems fortreating a cancer in a patient comprising a cytotoxic therapeutic agenthaving the characteristics of inhibiting the survival of a cancer cell,and an isolated population of cytotoxic immune cells, where thecytotoxic immune cells genetically modified to be resistant to thetherapeutic agent.

In embodiments of this aspect of the disclosure, the cytotoxic immunecells can be γδ T-cells.

In embodiments of this aspect of the disclosure, the population ofcytotoxic immune cells may comprise a heterologous nucleic acid sequenceoperably linked to a promoter, where the heterologous nucleic acidsequence encodes a polypeptide that when expressed in a cell confersresistance to the therapeutic agent to the cell.

In embodiments of this aspect of the disclosure, the therapeutic agentis a cytotoxic chemotherapeutic agent selected from the group consistingof: an alkylating agent, a metabolic antagonist, an antitumorantibiotic, and a plant-derived antitumor agent.

In some embodiments of this aspect of the disclosure, the therapeuticagent is selected from the group consisting of: TRIMETHOTRIXATE™ (TMTX),methotrixate (MTX), TEMOZOLOMIDE™, RALTRITREXED™,S-(4-Nitrobenzyl)-6-thioinosine (NBMPR), CAMPTOTHECIN™,6-benzylguanidine, and a therapeutic derivative of any thereof.

In certain embodiments of this aspect of the disclosure, the therapeuticagent is TRIMETHOTRIXATE™ or methotrixate, and the heterologous nucleicacid sequence encodes dihydrofolate reductase, or a derivative thereof.

In some embodiments of this aspect of the disclosure, the therapeuticagent is TEMOZOLOMIDE™, or a therapeutically agent derivative thereof,and the heterologous nucleic acid sequence encodes O⁶ methylguanine DNAmethyltransferase (MGMT), or a derivative thereof.

Still another aspect of the disclosure provides systems for treating aglioblastoma in a patient comprising a therapeutic agent having thecharacteristics of inhibiting the survival of a cancer cell and inducinga stress protein in the cancer cell, and an isolated population ofcytotoxic immune cells, wherein said cytotoxic immune cells are γδT-cells, and wherein said γδ T-cells have been genetically modified tobe resistant to the therapeutic agent.

In embodiments of this aspect of the disclosure, the population of γδT-cells comprises a heterologous nucleic acid sequence operably linkedto a promoter, wherein the heterologous nucleic acid sequence encodes apolypeptide that when expressed in a cell confers resistance to thetherapeutic agent to the cell.

In embodiments of this aspect of the disclosure, the therapeutic agentis a cytotoxic chemotherapeutic agent selected from the group consistingof: an alkylating agent, a metabolic antagonist, an antitumorantibiotic, and a plant-derived antitumor agent.

In some embodiments of this aspect of the disclosure, the therapeuticagent is selected from the group consisting of: TRIMETHOTRIXATE™ (TMTX),methotrixate (MTX), TEMOZOLOMIDE™, RALTRITREXED™,S-(4-Nitrobenzyl)-6-thioinosine (NBMPR), CAMPTOTHECIN™,6-benzylguanidine, and a therapeutic derivative of any thereof.

In some embodiments of this aspect of the disclosure, the therapeuticagent is TRIMETHOTRIXATE™ or methotrixate, and the heterologous nucleicacid sequence encodes dihydrofolate reductase, or a derivative thereof.

In other embodiments of this aspect of the disclosure, the therapeuticagent is TEMOZOLOMIDE™, or a therapeutically agent derivative thereof,and the heterologous nucleic acid sequence encodes O⁶ methylguanine DNAmethyltransferase (MGMT), or a derivative thereof.

It should be noted that ratios, concentrations, amounts, and othernumerical data may be expressed herein in a range format. It is to beunderstood that such a range format is used for convenience and brevity,and thus, should be interpreted in a flexible manner to include not onlythe numerical values explicitly recited as the limits of the range, butalso to include all the individual numerical values or sub-rangesencompassed within that range as if each numerical value and sub-rangeis explicitly recited. To illustrate, a concentration range of “about0.1% to about 5%” should be interpreted to include not only theexplicitly recited concentration of about 0.1 wt % to about 5 wt %, butalso include individual concentrations (e.g., 1%, 2%, 3%, and 4%) andthe sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within theindicated range. The term “about” can include ±1%, ±2%, ±3%, ±4%, ±5%,±6%, ±7%, ±8%, ±9%, or ±10%, or more of the numerical value(s) beingmodified. In addition, the phrase “about ‘x’ to ‘y’” includes “about ‘x’to about ‘y’”.

It should be emphasized that the above-described embodiments of thepresent disclosure are merely possible examples of implementations, andare merely set forth for a clear understanding of the principles of thisdisclosure. Many variations and modifications may be made to theabove-described embodiment(s) of the disclosure without departingsubstantially from the spirit and principles of the disclosure. All suchmodifications and variations are intended to be included herein withinthe scope of this disclosure and protected by the following claims.

EXAMPLES Example 1

Generation and titering of recombinant retrovirus: The cDNAs encodingfor human P140KMGMT and eGFP were PCR amplified using appropriateprimers (such as, for example, for: MGMT: Forward:AAACTGGAGCTGTCTGGCTGTGAA, Reverse: AAACTCTCCTGCTGGAACACTGGA; and DHFR:Forward: CATGGGAATTGGCAAGAATGGCGA, Reverse: TGACCAGGTTCTGTTTCCCTTCCA)

and inserted into the appropriate expression vector. The sequences,codon optimized for expression in mammalian cells, encoding MGMT andDHFR, are presented in FIG. 29.

A four plasmid system was used to generate recombinant SIV-lentivirus.Transient transduction was carried out in 293T producer cells usingmethods as detailed before (Cesano et al., (1998) Anticancer Res. 18:2289-2295, incorporated herein by reference in its entirety). The titersof virus encoding eGFP and P140KMGMT were determined by flow cytometryand real-time polymerase chain reaction (PCR) methods respectively(Cesano et al., (1998) Anticancer Res. 18: 2289-2295; McMillin et al,(2006) Hum. Gene Ther. 17: 798-806, incorporated herein by reference intheir entireties).

Example 2

Lentiviral transduction: Transductions of SIV based lentiviral particleswere performed by incubating cells with virus in media supplemented withpolybrene (8 mg/ml; Specialty Media, Phillipsburg, N.J.). Twenty-fourhours post transduction, virus-containing medium was replaced with freshmedium and transduced cells were cultured until reaching approximately70-90% confluency at which point cells were used for downstreamapplications.

Lentiviral transduction efficiencies of NK-92, TALL-104 and K562 cells:NK-92, TALL-104 and K562 cells were initially evaluated for theirtransduction efficiency using a self-inactivating (SIN) recombinant SWlentivirus, pseudo typed with the VSV-G envelope protein, encoding eGFP,the constructs of which are schematically shown in FIG. 25A.

The expression of eGFP in the lentiviral reporter construct was drivenby the murine stem cell virus promoter. To measure the transductionefficiencies, all cell lines were inoculated with an MOI of 40 and GFPfluorescence was analyzed by flow cytometry at 72 hrs aftertransduction. Transduction of each cell line resulted in robustexpressions of eGFP (as visualized by fluorescence microscopy) that werequantitated as 90%, 41% and 99% in the NK-92, TALL-104 and K562 celllines, respectively, as shown in FIGS. 25B-25D. Thus, high transductionefficiencies are achieved for both the NK-92 and K562 cell lines, andthe TALL-104 cell line exhibited moderate transduction efficiency.

Example 3

Survival curve analysis: The non-modified and P104KMGMT modified cellswere exposed 2 hours to 6-BG (25 μM) followed by exposure to increasingconcentrations of TMZ for 48 hrs. Cell viability was then accessed by astandard trypan blue exclusion method. All drugs were freshly preparedon the day of drug treatment.

To determine the effectiveness of drug resistant P140KMGMT, transducedand untransduced immunocompetent cells were incubated with 6-BG for 2hrs. TMZ was added at increasing concentrations, and the cells wereincubated for 48 hrs. Survival curves were generated with respect to thenon-transduced cells.

P140KMGMT transduced NK-92, TALL-104 and K562 cells were resistant tothe 6-BG/TMZ combination when compared to untransduced control cells, asshown in FIG. 26A). Such resistance was pronounced up to 200 μM TMZ, atwhich point nearly all modified cells survived the drug challenge.

The degrees of resistance achieved by genetic modification of each ofthe cell lines were measured by calculating the TMZ IC₅₀ value. The IC₅₀of 6-BG/TMZ based on 48 hrs exposure were 360±8 μM and 135±4 μM,respectively, in the gene-modified and unmodified NK-92 cells; 385±5 μMand 120±6 μM, respectively, in the gene-modified and unmodified TALL-104cells; and 550±8 μM and 170±10 μM, respectively, in the gene-modifiedand unmodified K562 cells. Each of the cell lines, therefore, showedapproximately a three-fold resistance to TMZ in a 48 hr viability assay.Similar resistance levels have been achieved in hematopoietic stem cellsand K562 cells, but using a 7-10 day survival assay (Gangadharan et al.,(2006) Blood. 107: 3859-3864). The choice of the present 48 hr assayperiod was based on downstream processing cytotoxic assays.

Example 4

Cytotoxicity assay: The cells, grown in the presence of 100 U/mLrecombinant human IL-2, were exposed to 25 μM 6-BG for 2 hrs followed bythe addition of 200 μMTMZ and incubating them overnight. To determinethe effector cell concentration that resulted in maximal killing of thetarget cells, 4,000 (T) cells were placed in 96-well plates and mixedwith the effector (E) cells (NK-92 or TALL-104) at E:T ratios of 2.5:1,5:1, and 10:1 (in triplicate) followed by a 4 hrs incubation. The amountof LDH released to the supernatant as a result of cytolysis of targetcells were measured in a lactate dehydrogenase (LDH) release assay(Roche Applied Science, Indianapolis, Ind.). Cytotoxicities wereexpressed as % cytotoxic activity of the effector cells according to thefollowing formula:

${\% \mspace{14mu} {cytotoxicity}} = {\frac{\begin{matrix}{\left( {{{Experimental}\mspace{14mu} {release}} - {{Spontaneous}\mspace{14mu} {release}_{effector}}} \right) -} \\{{Spontaneous}\mspace{14mu} {release}_{target}}\end{matrix}}{{{Maximum}\mspace{14mu} {release}_{target}} - {{Spontaneous}\mspace{14mu} {release}_{target}}} \times 100}$

To determine the effectiveness of the genetic modifications had on thecytotoxicities of the effector cells, the non-modified/gene-modifiedeffectors (E/Em) and target (T/Tm) cells were exposed to 6-BG/TMZ for 24hrs and cytotoxic assays were performed as detailed above.

Drug resistant variants of NK-92 and TALL-104 cell lines mediateeffective target cell killing: It has been reported that the NK-92 andTALL-104 cell lines can efficiently lyse the leukemic K562 cell line. Inthe present example, it was determined if genetic modification of theseimmune effector cell lines resulted in a change in their cytotoxicabilities towards the target cell line K562. Accordingly, gene-modifiedand non-modified effector cells were mixed with a fixed number of thetarget cells at various effector:target ratios of 2.5:1, 5:1 and 10:1.Killing effectiveness of each of the drug resistant effector cells werecompared with the unmodified control cells in a 4 hour cytotoxicityassay. When compared to the non-modified cells, both the gene-modifieddrug resistant immune effector cells, NK-92 and TALL-104 cells showedsimilar cytolytic activities toward the target cell line, as shown inFIG. 2B, thereby establishing that the genetic modifications imparted tothe NK-92 and TALL-104 transduced cell lines did not appear to affectcytotoxicity properties of the cells.

Retention of cytotoxic effectiveness of gene-modified immunocompetentcells after expansion in the presence of 200 μl TMZ. As shown in FIG.2B, the cytotoxicities of the drug selected gene-modified effector celllines NK-92 and TALL1-04, were similar to the cytotoxicities of thenon-selected gene-modified effector cells. These results show that thedrug resistant immunocompetent cell lines, after modification withP140KMGMT, retained their ability to efficiently lyze target cells.

Example 5

Generation of drug resistant effector and target cell lines bylentiviral transduction: The P140KMGMT cDNA sequence was inserted intothe SW expression vector by replacing the sequence encoding eGFP (asshown in FIG. 25A). Virus titers, determined using 293T cells astargets, were 10⁷-10⁸ TU/ml. Gene transfer into effector and targetcells was quantitated by real-time PCR amplification using genomic DNAisolated from cells transduced with the recombinant virus at an MOI of40.

P140KMGMT copy numbers for the transduced NK-92, TALL-104 and K562 cellswere determined to be 3±0.28, 1±0.14, and 4±0.41, respectively. MGMTmRNA levels were also measured in K562 cells to confirm thatgene-modified cells express increased levels of MGMT message. MGMT mRNAexpression was readily detected in the transduced K562 cells, while MGMTmRNA expression levels in untransduced K562 cells was below the linearrange of detection. Previous studies (Gangadharan et al., (2006) Blood.107: 3859-3864) also reported extremely low MGMT protein levels inwild-type K562 cells.

1-30. (canceled)
 31. An isolated composition comprising γδ T-cellsgenetically engineered to express a polypeptide that confers resistanceto a chemotherapy agent.
 32. An isolated composition comprising γδT-cells comprising a nucleic acid that encodes a polypeptide thatconfers resistance to a chemotherapy agent.
 33. The composition of claim31, wherein the a polypeptide that confers resistance to a chemotherapyagent is O⁶ methylguanine DNA methyltransferase (MGMT), a drug resistantvariant of dihydrofolate reductase (L22Y-DHFR), thymidylate synthase,multiple drug resistance-1 protein (MDR1).
 34. An isolated compositioncomprising natural killer cells wherein greater than about 50% of thenatural killer cells express a polypeptide that confers resistance to achemotherapy agent.
 35. An isolated composition comprising naturalkiller cells wherein greater than about 50% of the natural killer cellscomprise a nucleic acid that encodes a polypeptide that confersresistance to a chemotherapy agent.
 36. An isolated compositionconsisting essentially of natural killer cells comprising a nucleic acidthat encodes a polypeptide that confers resistance to a chemotherapyagent.
 37. The composition of claim 34, wherein the polypeptide thatconfers resistance to a chemotherapy agent is O⁶ methylguanine DNAmethyltransferase (MGMT), a drug resistant variant of dihydrofolatereductase (L22Y-DHFR), thymidylate synthase, or multiple drugresistance-1 protein (MDR1)